Hanahan competent E.coli
Andy Law Big Nose
Andy.Law at bbsrc.ac.uk
Wed Apr 19 08:19:44 EST 1995
In article <3n2mqg$3i4 at mserv1.dl.ac.uk>, LOGAND
<logand at msdos.montpellier.inra.fr> wrote:
> I am planning to prepare competent cells by the Hanahan method to use for
> the transformation of many ligation reactions (nested deletions prepared
> using the Pharmacia kit). I have never used this transformation protocol
> before, Sambrook et al. states that the number of cells in the culture
> should not exceed 1x10^8/ml and one should determine the relationship
> between OD and viable cell number/ml for the strain to be used. Well, I
> would love to be able to omit (or at least limit) this time consumming step
> - thus, can anyone give me a value for this using DH5a? I realise I will
> have to check/re-calculate this due to differences in spectrophotometers
> (light scattering etc) but at least I would have an approx. starting point.
We go for about 0.5-0.6 absorbance at 550nm using Hanahan RbCl method and
DH5a cells. It's not as critical as it's made out to be, although you have
to hit it on the up.
> Finally, do XL1 blue cells give worse or better transformation frequencies?
Depends on the construct, phase of the moon, time of day, direction of the
( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )
More information about the Methods